Microorganisms

ABSTRACT

MICROORGANISMS ARE ENTRAPPED IN A HYDROPHILIC ACRYLATE OR METHACRYLATE TO PROVIDE CONTROLLED RELEASE OR QUICK RELEASE OR TO PROVIDE A REGULATED TIME OF CONTACT WITH AN ENVIRONMENT ON WHICH THE MICROOGANISMS CAN ACT.

Oct. 23, 1973 A. GUTTAG 3,767,790

MICROORGANI SMS Filed Feb. 11, 1972 United States Patent 3,767,790MICROORGANISMS Alvin Guttag, Bethesda, Md., assignor to National PatentDevelopment Corporation, New York, N.Y.

Continuation-impart of abandoned application Ser. No.

832,938, June 13, 1969. This application Feb. 11, 1972,

Ser. No. 225,488

Int. Cl. A61k 23/00; C12k 1/10 U.S. Cl. 424-81 25 Claims ABSTRACT OF THEDISCLOSURE Microorganisms are entrapped in a hydrophilic acrylate ormethacrylate to provide controlled release or quick release or toprovide a regulated time of contact with an environment on which themicroorganisms can act.

This application is a continuation-in-part of Ser. No. 832,938 filedJune 13, 1969, now abandoned.

The present invention relates to the quick or controlled release ofliving microorganisms such as bacteria, molds, yeast and viruses or toproviding limited contact between the microorganism and an environmenton which it acts.

There are occasions when it is desirable to store living microorganismsso that they can be released or can act in an appropriate area and/or atan appropriate time. Thus, it may be desirable to protect bacteria orviruses until they can be implanted in the intestines or it may bedesirable to keep the microorganism dry or out of contact with air untiluse. It also is desirable on occasion to initiate the growth of onemicroorganism and later supplant the microorganism with a differentmicroorganism, e.g. in cheese making. Additionally it is frequentlydesirable to control the time of action of bacteria on a substrate.

It has not been found that such purposes can be accomplished byentrapping the microorganism in a hydrophilic acrylate or methacrylateester either in the form of a powder, tablet, pill or capsule. Theentrapped products are useful not only for therapeutic purposes but alsofor industrial purposes, eg in the manufacture of bread, cheese, citricacid, penicillin, oxytetracycline, streptomycin, erythromycin,bacitracin, gramicidin, tetracycline, tyrocidin, viomycin, kanamycin,aureomycin, beer, vinegar, alcohol, buttermilk, oxamycin,dihydrostreptomycin, benzyl penicillin, neomycin, lactic acid, butyricacid, gluconic acid, fumaric acid, butanediol, glycerol, propionic acid,propanediol, sulfite liquor, grape juice, sugar cane juice, sugar beetjuice.

In Shepherd application Ser. No. 550,453, filed May 16, 1966, and nowabandoned, and commonly assigned there is described a petri dishcontaining nutrients for bacteria in which the dish is made of ahydrophilic hydrogel in accordance with Wichterle U.S. Patents Nos.2,976,576 and 3,220,960. The nutrients in solution in a solvent areabsorbed within the hydrogel which is then dried and can be stored. Nobacteria are in the dish at this stage. When it is desired to provide anutrient solution, e.g. for growth of bacteria, the dish is removed fromstorage, solvent added in order to leach the solid nutrient out of thepolymer and to recreate the nutrient solution.

The Shepherd disclosure is distinguished from because Shepherd does notentrap or coat bacteria or other living microorganisms in thehydrophilic polymer. Instead Shepherd entraps nutrient materials whichare then leached out of the polymer.

When a water soluble or organic solvent soluble hydrophilic polymer isemployed the microorganism can be set free by dissolving the polymer inwater or organic solvent. The microorganisms are too large to be leachedfrom the polymer by water or organic solvent.

In another aspect of this invention microorganisms are provided in theforms of reusable systems and systems which can be employed forcontrolled time periods displaying durable microbial activity ofincreased stability. It has been found that such devices can be preparedby immobilizing active microorganisms, e.g. bacteria and yeast in ahydrophilic polymeric matrix by chemical methods or physical entrapment.Such immobilized microorganism systems can be prepared by a variety ofmethods which include:

(1) First dissolving or suspending the microorganisms prior topolymerization in the monomeric mixture including a cross-linking agentand then proceeding with the polymerization, resulting in a cross-linkedinsoluble polymeric gel lattice. The macromolecular structure of such alattice can be controlled by varying the nature and the concentration ofthe monomeric moieties so that the microorganism will be retained in thegel matrix whereas molecules of substrate and reaction product, ofsmaller size will be able to move freely in the polymeric network.

(2) First mixing the microorganisms with an aqueous or organic solutionof the polymer, and then cross-linking the polymeric mixture to renderthe entrapping matrix insoluble.

(3) First absorbing a suspension of the microorganisms in a porousstructure such as natural and synthetic foams, porous organic andinorganic materials (e.g. foamed polymethane), e. g. (toluenediisocyanate-polytetramethylene glycol), foamed urea-formaldehyde,foamed phenol-formaldehyde, foamed polystyrene, foamed polyethylene,foamed polypropylene, foamed natural rubber, foamed butadiene-styrenecopolymer, foamed epoxy resin (bisphenol A-epichlorhydrin), activatedcarbon, porous glass, porous metal, activated alumina, silica gel,foamed or sponge hydroxyethyl methacrylate polymers and then removingthe solvent under reduced pressure.

All the devices described above can then be given an additional coatingof hydrophilic polymer. This external membrane is useful in prolongingthe shelf-life of the microorganisms.

The thickness of this external membrane can be designed to determine therate of difiusion of the substrate to the vicinity of the microorganismas well as the rate of diffusion of the products out of the reactionsite. The thickness can be 5 microns to 1 mm., preferably not over 500microns, most preferably 10-50 microns. The chemical composition of thisexternal membrane, and its macromolecular structure in the swollen statein contact with the substrate medium, will only allow dissolvedmolecules below a certain range of molecular weights to dilfuse throughit, i.e. molecules of lower molecular weight than the microorganisms,e.g. usually up to a molecular weight of 10,000, but if desired, thehydrophilic polymer can be designed to permit molecules up to 50,000pass through so that proteins such as insulin, zein, gliadin andlactoglobulin for example will pass through.

The microorganism containing device if made from a water insolublehydrophilic polymer preferably either contains a layer of entrappedenzyme having a coating of hydrophilic polymer or is in the form of ahollow tube having a strengthening fiber therein or is in the form of acylindrical plug in an otherwise hollow cylinder.

The said microorganisms containing devices can be shaped as hard beads,coarse or fine powders, rods, tubes, multilayer membranes, films,fibers, hollow fibers, pouches, capsules. They can also be applied onthe surface of solid substrates of any shape by adopted coating methods.

In the devices of the invention the microganisms remain permanentlyentrapped and they do not leach out nor does the device dissolve to setthem free except in those cases when a water soluble or organic solventsoluble hydrophilic polymer is employed and then water or organicsolvent is employed to set free the microorganism.

The said systems are sometimes prepared in the presence of water,bufiFer solution, or solvent systems that do not destroy themicroorganisms, however, they can be subsequently totally dehydarted toprovide an easier method of storage, with an appreciable increase inshelf life. Moreover, after use, the devices can be kept for furtherre-use over periods of months without appreciable loss of activity ifthey are dried at temperatures which do not kill the microorganisms.

Polymer matrixes are preferably made from a hydrophilic monomer which isa hydroxy lower alkyl acrylate or methacrylate, or hydroxy lower alkoxylower alkyl acrylate or methacrylate, e.g., Z-hydroxyethyl acrylate,Z-hydroxyethyl methacrylate, diethylene glycol monoacrylate, diethyleneglycol monomethacrylate, 2-hydroxypropyl acrylate, Z-hydroxypropylmethacrylate, 3-hydroxypropyl acrylate, 3-hydropropyl methacrylate anddipropylene glycol monomethacrylate. The preferred monomers forpreparing the matrixes are hydroxyalkyl acrylates and methacrylates,most preferably, 2-hydroxyethyl methacrylate. The polymers produced fromslurries of monomers are organic solvent soluble, e.g. alcohol soluble,but water insoluble. They can be prepared for example as shown inShepherd Patent 3,618,213 e.g. Example 36a, or Chromacek Patent3,575,946.

The hydroxyalkyl acrylate or methacrylate less preferably can also bereplaced in part by vinyl pyrrolidone, acrylamide, methacrylamide,N-propyl acrylamide, N-isopropyl methacrylamide, N-methylacrylamide,N-rnethylmethacrylamide, N-methylol acrylamide and N-methylolmethacrylamide, N-Z-hydroxyethyl acrylamide, N-Z-hydroxyethylmethacrylamide. However, these monomers usually form water solublehomopolymers and hence they require the presence of a cross-linkingagent or copolymerization with a suificient amount of the hydroxyalkylacrylates and methacrylates to render the copolymers water insoluble foruses where the polymer is not to be dissolved.

Other ethylenically unsaturated monomers can be used in conjunction withthe above monomers or copolymers to constitute hydrophilic polymericmatrixes suitable for the entrapment of enzymes. They include neutralmonomers such as acrylonitrile, methacrylonitrile, vinyl acetate, alkylacrylates and methacrylates, alkoxyalkyl acrylates and methacrylates.

Examples of alkyl acrylates and methacrylates include methyl acrylate,ethyl acrylate, butyl acrylate, 2-ethylhexyl acrylate, methylmethacrylate and butyl methacrylates. Examples of suitable alkoxyalkylacrylates and methacrylates are methoxyethyl acrylate, methoxyethylmethacrylate, ethoxyethyl acrylate, ethoxyethyl methacrylate,propoxyethyl acrylate, butoxyethyl methacrylate, methoxypropyl acrylate,ethoxypropyl methacrylate. These comonomers when used in an amountpreferably not higher than 50 percent (and usually between 0.5 and 20%)of the monomeric mixture contribute to improve the mechanical prooertiesof the gel, They should not be used in an amount to impair thehydrophilic nature of the polymer. Other vinyl monomers bearingionizable functional groups can be copolyrnerized with the hydroxyalkylacrylates or methacrylates to constitute ionogenic matrixes which can beuseful when a basic or acidic environment is required for the stabilityor the optimum activity of enzymes. They include acidic type monomerssuch as acrylic acid, methacrylic acid, maleic acid, fumaric acid,itaconic acid, aconitic acid, cinnamic acid, crotonic acid, tricarbyllicacid, propiolic acid, citraconic acid, vinyl sulfonic acid,p-vinylbenzenesulfonic acid, partial esters such as mono-Z-hydroxyethylitaconate, mono-Z-hydroxypropyl citraconate, mono-Z-hydroxyethylmaleate, mono Z-hydroxypropyl fumarate, monomethyl itaconate, monoethylitaconate, monomethyl Cellosolve itaconate (methyl Cellosolve is themonoethyl ether of diethylene glycol), monomethyl Cellosolve maleate,mono-Z-hydroxyethyl aconitate.

They also include basic type monomers such as aminoethyl methacrylate,dimethyl aminoethyl methacrylate, monomethylaminoethyl methacrylate,t-butylaminoethyl methacrylate, p-aminostyrene, o-aminostyrene, Z-amino-4-vinyltoluene, diethylaminoethyl acrylate, dimethylaminoethyl acrylate,t-butylaminoethyl acrylate, piperidinoethyl acrylate, piperidinoethylmethacrylate, morpholinoethyl acrylate, morpholinoethyl methacrylate,Z-vinyl pyridine, 3-vinyl pyridine, 4-vinyl pyridine, Z-ethyl-S-vinylpyridine, dimethylaminopropyl acrylate, dimethylamino propylmethacrylate, dipropylaminoethyl acrylate, dimethylaminoethyl vinylether, dimethylaminoethyl vinyl sulfide, diethylaminoethyl vinyl ether,aminoethyl vinyl ether, 2-pyrrolidinoethyl methacrylate,3-(dimethylaminoethyl -hydroxypropyl acrylate, 3- dimethylaminoethylZ-hydroxypropyl methacrylate, 2-aminoethyl acrylate, 2- aminoethylmethacrylate. The alkylaminoethyl acrylates and methacrylates arepreferred in this group. These ionogenic monomers should not be used insufiicient amounts to render the hydroxyalkyl acrylates or methacrylateswater soluble unless a water soluble polymer is desired. In particularcases, the most suitable matrix for the entrapment of the bacteria maybe composed of multipolymers prepared from a mixture of 3, 4 or more ofthe above monomers. These monomers are usually used in an amount of 0.1to 20%, preferably 1 to 15% of the total monomers.

In making a matrix suitable for bacteria entrapment it is oftennecessary to render said matrix insoluble in water or organic solvents.This is done by sparingly cross-linking the entrapping polymer.Preferably, the cross-linking agent is added in an amount of 1 to 10%,most preferably not over 2.0% or 2.5%, although from 0.05 to 15% or even20% of cross-linking agents can be used. Cross-linking renders theotherwise organic solvent soluble or water soluble polymers insoluble,although it does not impair the hydrophilic properties. It is obviousthat a non cross-linked organic solvent soluble polymeric system may beused when the substrate or the non-aqueous liquid used to dilute thesubstrate, do not constitute a solvent for the entrapping matrix when itis desired to retain the microorganisms in the polymer matrix.

Typical examples of cross-linking agents include ethylene glycoldiacrylate, ethylene glycol dimethacrylate, 1,4- butylenedimethacrylate, diethylene glycol dimethacrylate, propylene glycoldimethacrylate, diethylene glycol dimethacrylate, dipropylene glycoldimethacrylate, diethylene glycol diacrylate, dipropylene glycoldiacrylate, divinyl benzene, divinyl toluene, diallyl tartrate, allylpyruvate, allyl malate, divinyl tartrate, triallyl melamine, N,N'-methylene bisacrylarnide, diallyl maleate, divinyl ether, diallylmonoethylene glycol citrate, ethylene glycol vinyl citrate, allyl vinylmaleate, diallyl itaconate, ethylene glycol diester of itaconic acid,divinyl sulfone, hexahydro-l,3,5- triacryltriazine, triallyl phosphite,diallyl ether of benzene phosphonic acid, polyester of maleic anhydrideWith triethylene glycol, diallyl aconitate, divinyl citraconate, diallylfumarate, ammonium dichromate. Of course crosslinking agents andmonomers which form polymers toxic to the specific microorganisms shouldnot be employed.

In order to make polymers which are not only hydrophilic but also watersoluble, e.g. for use to entrap yeast for bread manufacture, there canbe employed copolymers of the hydroxyethyl or hydroxypropyl acrylatewith 0.5 to 20% of an ammonium or alkali metal salt of an ethylenicallyunsaturated carboxylic acid or the strong acid salt of an ethylenicallyunsaturated amine. Thus, there can be used ammonium, potassium andsodium salts of acrylic acid, methacrylic acid, maleic acid, mono-Z-hydroxyethyl itaconic and mono-Z-hydroxypropyl maleate as well ashydrochloric, hydrobromic, sulfuric, ni-

tric and phosphoric acid salts of dimethylaminoethyl methacrylate,t-butylaminoethyl methacrylate, p-aminostyrene, dimethylaminoethylacrylate and triethanolamine monomethacrylate or there can be usedsimilar amounts of vinyl pyrrolidone, acrylamide, methacrylamide or thelike.

Polymerization can be carried out by various procedures. Thus thepolymer can be formed as a casting syrup and then cured. Alternatively,the hydrophilic polymers are prepared by solution polymerization or bysuspension polymerization of the hydrophilic monomer, including thecross-linking agent (if employed) and stopping the polymerization whenthe polymer formed will precipitate in water (if a water solubilizingmonomer is not included) but is still soluble in highly polar organicsolvents such as alcohols, glycols, and glycol ethers. Examples ofsuitable solvents are ethyl alcohol, methyl alcohol, isopropyl alcohol,ethylene glycol, propylene glycol, diethylene glycol, dipropyleneglycol, monomethyl ether of ethylene glycol, dimethyl formamide,dimethyl sulfoxide and tetrahydrofurane. Polymerization to form solventsoluble polymers can be carried out for example as in Shepherd Patent3,618,213 or Chromacek Patent 3,575,946.

Suspension polymerization is carried out in a nonpolar medium such assilicone oil, mineral oil, xylene, toluene, e.g. as in Examples 36a, 36band 360 of said Shepherd patent.

The soluble polymer formed by solution or suspension process is thendissolved in the appropriate solvent as indicated above and can beadmixed with the microorganism. The solvent selected of course shouldnot be toxic to the organism. The solvent is then removed and, ifnecessary, a process leading to a cross-linked structure carried out attemperatures preferably below 40 C. resulting in thin insoluble filmsentrapping the active enzyme. Temperatures of 5 C., or 0 C. up to C. arefrequently employed to insure that there is not a premature growth ofthe microorganism. Temperatures of 20 C., 25 C. and 37 C. can be used(or even higher with thermophilic organisms).

When the microorganism containing device is formed by direct entrapmentin a cross-linked matrix, a useful method of preparing the polymermicroorganism matrix consists in polymerizing a casting solutioncontaining monomer or monomers, cross-linking agent if employed, andcatalyst, in which microorganisms have been suspended in presence ofvariable amounts of distilled Water, aqueous buffer solutions or organicsolvents. Proper pH, i.e. the preferred pH for activity of the specificmicroorganism, enhances the subsequent growth of the microorganism. Theamount of water, aqueous buffer or organic solvent can vary from 0 to100% of the weight of the monomers and can even be more, e.g. 1000% or1500% of such weight. The resulting casting solution is allowed topolymerize in molds of predetermined shapes so that the immobilizedmicroorganism matrix appears as films, rods, or tubes. Suchpolymerizations are generally carried out at temperatures below 40 C. aspreviously indicated. If anhydrous conditions are not used nutrientsshould preferably be avoided in the polymerization medium or the productdried.

Typical organic solvents include alcohols such as methyl alcohol, ethylalcohol, propyl alcohol, isopropyl alcohol monomethyl ether ofdiethylene glycol, monoethyl ether of diethylene glycol, monoethyl etherof ethylene glycol, monoethyl ether of ethylene glycol, dioxane,dioxane-water mixture, alcohol-water mixture (e.g. 95% alcohol),pyridine, dimethyl formamide, dimethyl sulfoxide, tetrahydrofur'furylalcohol, ethylene glycol, propylene glycol, formamide, cyclohexanol,glycerol, toluene, xylene, benzene, triethylene glycol, t-butanol.

Rods or films can also be ground into coarse or fine powders suitablefor filling columns, cartridges, or permeable bags. A final surfacecoating with polymer solution or polymerizable monomer may then be addedwhen necessary to insure that no microorganism moiety is directlyexposed to the substrate. The solvent is either removed by evaporationor otherwise until a solid coating, e.g. a gel is formed or themonomerized to form a solid coating.

Strengthening materials such as woven glass fibers, Dacron (polyethyleneterephthalate) nylon (e.g. nylon 6, nylon 6,6, nylon 6,10),polyacrylonitrile fibers, vinyl chloride polymer fibers, and the like,woven gold, platinum, stainless steel thread and the like, in variousmil and mesh sizes, can be present in the mold prior to polymerizing sothat the strengthening material is completely encased in the castingsolution. The resulting microorganisms-containing, membranes, rods andtubes are then able to sustain higher tension pressure and roughhandling.

Another typical system for entrapping microorganisms can be prepared ashard microporous beads of various sizes, which can be used in a columnthrough which the substrate flows, or in a vessel mixed with thesubstrate and easily recoverable after the reaction.

Such bead-like devices composed of a cross-linked hydrophilic matrixentrapping microorganism (a single type of microorganism as well as amixture of various microorganisms for use in some reactions) can beprepared when a suspension polymerization is carried out on non polarmedia such as silicone oil, mineral oil, parafin oil, oxylene, benzene,toluene or in presence of a high molecular weight poly-isobutylene. Themicroorganisms are suspended in the monomeric phase in presence ofeither a small amount of water (with or without buffer) or an organicsolvent, the catalyst system, the cross-linking agent (0.05 to 20%usually 12.5% of monomer weight). The polymerization is carried outunder constant stirring rate and controlled temperature (5 to +40 C. forexample, usually 0 to 25 C.). At the end of the polymerization time,porous, hard, spherical beads in which the microorganisms are entrapped,are collected, and if desired, rapidly washed in an appropriate solvent,and then, if desired, leached in water to remove residual monomer,residual catalyst, and unbound microorganisms. Beads of various size canbe obtained by varying the ratio of the monomeric phase to thesuspending phase, under adequate stirring. A coating of the hydrophilicpolymer can then be applied to the beads in any desired fashion.

As catalysts for carrying out the polymerization, there is usuallyemployed a free radical catalyst in the range of 0.05 to 1% of thepolymerizable monomer. The preferred amount of catalyst is 0.1 to 0.5percent of the monomer.

Polymerization can be carried out at 20 to 150 C., usually at 40 to C.for the preparation of the matrix used for solution entrapment ofmicroorganisms, or preferably at 5 to 40 C., usually at 0 to 25 C., whenthe microorganisms are present in the polymerizable mixture. The lowertemperatures are employed when microorganisms are present to avoideither permature growth or killing of the microorganisms.

Typical catalysts used for the polymerization of the matrix includet-butyl-peroctoate, benzoyl peroxide, iso propyl percarbonate, methylethyl ketone peroxide, cumene hydroperoxide,1,3-bis-(t-butylperoxyisopropyl) benzene and dicumyl peroxide. Anothergroup of catalysts useful mainly for low temperature polymerizationincludes redox systems such as potassium persulfate-riboflavine,potassium persulfate-sodium bisulfite, hydrogen peroxide-divalent iron.Various compounds such as N,N,N,N'-tetramethylethylenediamine can beused to accelerate the effect of the catalysts. Irradiation, e.g., byultraviolet light of gamma rays can also be employed to catalyze thepolymerization. The catalyst is not critical and any of thoseconventional in the art can be employed.

The polymer formed in the suspension polymerization or obtained from thecasting syrup, if desired, can be dissolved in water or the appropriatesolvent as indicated above providing the solvent is not toxic to themicroorganism and can be admixed with any suitable substrate containingthe microorganisms, e.g. agar, the solvent removed and, if necessary,the olymerization completed. Alternatively, the solvent containingpolymer solution can be coated on microorganisms entrapped in solventinsoluble hydrophilic polymer and the solvent removed to form a film orcoating for the entrapped material.

When the casting syrup is employed, polymerization can be carried outuntil a solid is formed with or without the microorganism entrappedtherein. If the cast material is to be employed as a coating for themicroorganisms rather than merely being in admixture therewith, thecentral portion of the casting is hollow and an entrance to the hollowportion is provided, either by the shape of the initial casting orsimply by cutting a hole therein, the microorganisms inserted, and theaperture closed with more partially cured polymer followed by completionof the cure.

The casting syrup can be cured to form products which exist in a solidstate, e.g. rigid state, and can be swollen. The cross-linked polymerobtained from the cured liquids has reversible fluid absorptionproperties, the ability to retain its shape in a fluid absorption mediaand to elastically recover its shape after deformation. The watersoluble polymers will simply dissolve in water to expose themicroorganisms and render them available. When insoluble polymers areemployed the microorganisms are retained in the polymer matrix and theliquid system on which the microorganisms are to act must be absorbed bythe matrix.

Polymeric powders prepared by any of the methods set forth above as Wellas by disintegrating a polymer produced in the form of a foam are mixedwith the desired microorganisms with the aid of an appropriate solventif necessary, and the mixture placed on a mechanical roller so that thematerials can be intimately mixed. The mixture is dried by airevaporation or gentle heat or by freeze drying. Upon evaporation of thesolvent, e.g. water, the microorganism substance is retained by thepowder. The powder can be formed into pills, tablets or capsules ifdesired. Due to its extreme hydrophilicity and because the hydrophilicpolymer of this invention has reversible fluid adsorption properties,the powders can be reconstituted in a biological system so that themicroorganism can be reached by the biological fluid which passesthrough the polymer matrix at a steady rate (except in the case of thewater soluble polymers in which event the entrapping polymer is simplydissolved).

As microorganisms, for example, there can be used Streptococcus lactis,Saccharomyces cerevisiae, Lactobaccillus delbrzteckii, AspergillusNiger, Acetobacrer rances, Bacterium curvum, Laclobacillus bulgaricus,Lactobacillus cascf. Lactobacillus lcichmamzii, Cirromyccs pfefierianus,Penicillin aremzrium, Rhizopus nigricans, Penicillin noratum,Streptomyces griseus, Strepromyces citrovorus, Penicillin roqueforti,Lactobacilltts cucumcms, Influenza virus e.g. type A, strain PR-8),Laczobacillus bifidus, Lactobacillus acidophflus, Escherichia coli,Saccharomyces cerevisz'ae, Saccharomyces anamensis, Rhizopus nigricans,Aspergillus oryzae, Saccharomyccs ellipsoideus, Saccharomycespastorianus, Clostridium bulyricum, Streptococcus lacris,Propionibaczerium freudenreiclzii, Srreptomyces rimosus formaparomycz'nus, Aczinomyces griseus, Streptomyces kanamyceticus, Srrepfomyces humidus, adenovirus type 3 (Camp Lejeune 15520 strain),adenovirus type 7, attenuated poliomyelitis virus Type I (SM strain) orType II (TN strain) or Type III (Fox strain) (see Patent No. 2,946,724),poliomyelitis virus of the MEI-1 strain of the Lansing type attenuatedby at least 119 serial passages in suckling hamsters (see Patent No.3,125,489), live attenuated poliomyelitis virus of Sabins Type I, II orIII, infectious bovine rhinotracheitis virus (I.B.R. virus) attenuatedas set forth in Patent No. 3,048,524, I.B.R. virus (Ithaca strain)attenuated by 50 passages in lamb renal cells, attenuated live measlesvirus (Edmonston strain) attenuated as set forth in Patent No.3,133,861Example 2, attenuated B.C.G. (bovine tubercle Bacillus),attenuated live rabies virus (Flury strain) further cultured in chickembryo tissue culture as set forth in Patent No. 3,255,080, attenuatedchick embryo modified canine distemper virus, avirulent livingduck-embryo modified infectious canine hepatitis virus (see Patent No.3,153,474), attenuated avian pneumoencaphalitis virus (see Patent No.3,155, 588), avirulent Bacillus anthracis strains ATCC 14185, ATCC 14186and ATCC 14187, attenuated hog cholera virus (porcine origin or rabbitorigin) or tissue culture origin (see Patent No. 3,226,296), attenuatedvirus diarrhea virus (Oregon 024V strain) attenuated as set forth inPatent No. 3,293,129, attenuated panleukemia virus (see Patent No.3,293,130), attenuated Salmonella dublin (strain ATCC 15480), attenuatedSalmonella gallinarum, noninfectious rinderpest virus (see Patent No.2,756,176), rumen microorganisms, Newcastle virus 9251 strain)attenuated by passages in embryonated chicken eggs, see strain ofinfluenza inactivated as set forth in Patent No. 3,058,894, mixed caninedistemper virus, rabies virus and infectious hepatitis virus or thelike.

The combination of the hydrophilic polymer and entrapped microorganismif used as a medicine can be taken orally or implanted parenterally,enterally, or subcutaneously or can be shot into the body (in eitherveterinary medicine or human therapy) in capsule form with a bolus gunor other conventional technique.

The novel formulation of this invention comprises a. dosage unitcombination capable of releasing the microorganisms in the case of thewater soluble polymer, immediately. The rate of release for the mostpart will be determined by the ratio of the hydrophilic polymer to thematerial containing the microorganism, by the sequence or thickness ofthe coatings which are employed, or by the presence of one or moreblocking layers. Blocking layers used in the invention may be any ofthose ingestible materials conventionally employed including waxes suchas beeswax, carnauba wax, Japan wax, paraffin, bayberry wax, higherfatty acids, such as oleic acid, palmitic acid and stearic acid, estersof such higher fatty acids such as glyceryl tristearate, cetylpalmitate, diglycol stearate, glyceryl myristate, triethylene glycolmonostearate, higher fatty alcohols such as cetyl alcohol and stearylalcohol, and high molecular weight polyethylene glycols such as theCarbowaxes, polyethylene glycol mono-stearate, polyethylene glycoldistearate, polyoxyethylene stearate, glyceryl monostearates andmixtures thereof.

The blocking layer or the coating layer can be 0.1 to 5 mils thick toretard the availability of the inner microorganisms. The thickness canbe chosen for any desired time delay.

Unless indicated, all parts and percentages are by weight.

The invention can be employed for example in preparmg ethyl alcohol fromaqueous solutions of sucrose, maltose, fructose, invert sugar orraflinose (e, g. a 15% sucrose solution) using ahydroxyethylmethacrylate polymer (HEMA polymer) containing entrappedSaccharomyces cerevisz'ae; or from glucose or fructose (eg. as a 10%aqueous solution) with HEMA polymer containing entrapped Saccharomycesellipsoz'deus. Acetic acid can be prepared from aqueous ethyl alcohol(eg. 10% alcohol) using entrapped Bacterium curvnm or B. orl'eanese orAcetobacter rancens. Lactic acid can be prepared from aqueous lactose,maltose, glucose, sucrose or dextrins or molasses (e.g. 10% lactose)using entrapped Lactobacillus delbrueckii, L. bulgaricus or L. casei.Butyric acid can be prepared from aqueous sucrose or molasses (e.g. 10%

aqueous molasses) using entrapped Clostridium butyricum. Citric acid canalso be prepared from sucrose or molasses (e.g. 12% aqueous sucrose)using entrapped Citromyces pfefierianus. Gluconic acid can be preparedfrom glucose (e.g. 10% aqueous glucose) using entrapped Aspergillusniger. Fumaric acid can be prepared from glucose using entrappedRhizopus nigricans. Antibiotics can also be prepared using conventionalorganisms, e.g. penicillin using entrapped Pe nic 'llium notatum;streptomycin using Streptomyces griseus, aureomycln using Streptomycesaureofaciens and chloromycetin using Streptomyces venezuelae. Theconventional auxiliary nutrients can also be added to either the liquidpassed through the entrapped microorganism or, if the auxiliary agentsare water insoluble, they can be included with the entrappedmicroorganisms.

When a water insoluble hydrophilic polymer is employed as the entrappingagent in making the products set forth above, it is preferably a polymerof HEMA, usually containing about 0.2 to 0.3% ethylene glycoldimethacrylate as a cross-linking agent. The time of contact of theliquid passing through the entrapped microorganism is usually 12-24hours but the time can be shorter e.g. 4 hours, or longer, e.g. 36, 48or 96 hours.

If the hydrophilic polymer containing entrapped microorganisms are inthe form of beads or other small particles, they can be convenientlyretained in a tube between two screens so that the nutrient containingliquid, e.g. aqueous sucrose, can pass through the screens but the beadsbe retained in order to limit the time of action. The preferred methodof controlling the time of contact, however, is that shown in FIG. 3 ofthe drawings.

The invention will be understood best in connection with the drawingswherein:

FIG. 1 is a cross sectional view of a tablet, pill or capsule or pelletaccording to the invention.

FIG. 2 is a cross sectional view of another form of tablet or pill;

FIG. 3 is a cross sectional view of a tube having entrappedmicroorganisms according to the invention;

FIG. 4 is a cross sectional view illustrating a device for storing aproduct of the invention; and

FIG. 5 is a cross sectional view of another embodiment of the invention.

Referring more specifically to FIG. 1 of the invention there is provideda capsule (pill or tablet) 2 in capsule form comprising microorganisms 4in dry form (e.g. yeast, Lactobacillus bifidus, Penicillin roqueforli,attenuated I.B.R. virus, etc.) as a core with a coat 6 of thehydrophilic polymer previously described, e.g. 2-hydroxyethylmethacrylate-ethylene glycol dimethacrylate (100: 0.2). When takenorally or implanted in a biological system, the hydrophilic coatingallows body fluids or industrial fluid, e.g. aqueous sucrose topenetrate at a predetermined rate and contact the microorganisms for apredetermined period of time. The coating can have a thickness, for.example, of 1.0 mil. If the coating is of a water soluble polymer, itwill dissolve to set free the microorganisms.

As shown in FIG. 2 a capsule (pill or tablet) 8 has a core comprisingmicroorganisms 10 entrapped on the hydrophilic hydroxyethyl methacrylatepolymer 12 having an enteric coating 14, e.g. of cellulose acetate,shellac, methyl cellulose, polyethylene glycol 6000, etc.

On occasion it is important to store the microorganisms, e.g. underanaerobic conditions. This can be done according to the invention asshown in FIG. 4 by placing the microorganisms entrapped in thehydrophilic polymer (e.g. Z-hydroxyethyl methacrylate-ethylene glycoldimethacrylate copolymer (100:O.4)) as particles 16 in glass bottle 18having a conventional rubber stopper 20 having an overall coat 22 ofSaran (e.g. vinylidene chloride-acrylonitrile copolymer (80:20)). Thebottle can either be evacuated or filled with a dry nitrogen, argon orhelium atmosphere prior to sealing. Bottles of this type are shown inFrench Pat. No. 1,266,294. If desired, the bottle can also have aconventional aluminum securing ring around the rubber stopper.Alternatively, glass containers with ground glass stoppers can be usedto maintain a nitrogen atmosphere or the granules (or capsules or pills)can simply be wrapped in metal foil impervious to the atmosphere.

The container 18 can also be used to provide a holder for reaction. Thusif the microorganisms entrapped are Aspergillus niger, there can beadded an aqueous solution (e.g. 15%) of glucose (at a pH of 3.5 toproduce gluconic acid or a pH of 2.0 to produce citric acid). Thesolution is then allowed to remain in contact with the beads for 48hours at room temperature, drained off and the particles dried andstored for subsequent use. The gluconic acid (or citric acid) formed isthen recovered from the aqueous liquid in conventional fashion from thedrainedoff product.

As shown in FIG. 3 there is provided a cylindrical tube 24 of any inertmaterial, e.g. nickel or polyethylene, having hollow sections 26 and 28joined by an intermediate section 30. The intermediate section 30comprises an inner cast copolymer of hydroxyethyl methacrylateethyleneglycol dimethacrylate (10020.3) 32 having entrapped therein any suitablemicroorganism, e.g. Asper gillus niger or yeast or Penicillium notatum.Completely encasing the cast polymer are top and bottom membranes 34 and36 of hydroxyethyl methacrylate-ethylene glycol dimethacrylate (:3). Themembranes are permeable to aqueous solutions and are impermeable tomicroorganisms. An appropriate aqueous solution, e.g. a 10% glucosesolution at pH 3.5 is passed through tube 24 from section 26 throughmembrane containing section 30 and then to section 28. Gluconic acid isformed during the passage through section 30, e.g. during a period of 4hours and the gluconic acid containing solution is withdrawn from tubesection 28 and recovered. The time it takes the solution to pass throughsection 30 of the tube can be regulated to any desired time simply byvarying the thickness of the membranes 34 and 36 and the thickness ofthe cast layer 32. If desired either or both of membranes 34 and 36 canbe omitted but their use is preferred to insure stability of theentrapped microorganisms in storage.

In place of a tube such as that shown in FIG. 3, there can be employed atube 40 as disclosed in FIG. 5 composed of a hydrophilichydroxyethylmethacrylate polymer wall 44, preferably having areinforcing sheathing, e.g. of Dacron mesh 46. The tube has entrappedtherein the microorganism. An aqueous solution, e.g. 10%sucrose,ispassed through the lumen 47 of the tube with the result that thesolution diffuses into the microorganism entrapped tube. If themicroorganism is Aspergillus niger and the aqueous sucrose solution hasa pH of 2.0, there is diffused out of the outer surface 48 of theexternal portion of the tube an aqueous solution of citric acid whichcan be collected in any desired manner. To preserve the shelf life ofthe bacteria (or other microorganism) the tube 40 can have inner andouter coatings of a hydrophilic water insoluble HEMA polymer. To controlthe rate at which an aqueous solution in the lumen of the tube can reachthe microorganism, there can be used an inner coating of a hydrophilicwater insoluble HEMA polymer.

The following examples will further illustrate the invention.

EXAMPLE 1 2-hydroxyethyl methacrylate is stirred with 0.15 gram per 100grams of methacrylate of isopropyl percarbonate in an anaerobicatmosphere at ambient temperature. Ethylene glycol dimethacrylate in theconcentration of 0.1 gram per 100 grams of 2-hydroxyethyl methacrylateis added. Then a lyophilized mixture (prepared by lyophilizing 30 gramsof moist Lactobacillus acidophilus 11 (containing grams of bacteria and75 grams of water), grams of skim milk powder, 5 grams lactose and 0.5gram xylose) containing L. acidophilus was added in an amount of 10grams per 100 grams of methacrylate to provide a casting syrup.

The casting syrup was polymerized to a solid by heating to 40 C. andadding 0.3 gram of further isopropyl percabonate. This product wasuseful as such or as a core for a capsule having an outer entericcoating of cellulose acetate hydrogen phthalate, methyl cellulose or thelike. There can be employed in place of the L. acidophilus, E. coli,Saccharomyces cerevisiae, Acetobacter rances, attenuated, I.B.R. virus,attenuated hog cholera virus or any of the other microorganismsmentioned supra.

EXAMPLE 2 Distilled Z-hydroxy ethyl methacrylate (100 g.) is stirredwith 0.1 g. tertiary butyl peroctoate in an anaerobic atmosphere at25-70 C. for -40 minutes. The resultant mixture is cooled to C. andtertiary butyl peroctoate added so as to make the total concentration oftertiary butyl peroctoate in the system 0.2/100 grams of 2-hydroxy ethylmethacrylate. Ethylene glycol dirnethacrylate, in a concentration of 0.2g./100 g. of Z-hydroxy ethyl methacrylate is added at the same time asthe catalyst concentration is brought up to the theoretical content.

100 g. of the resulting syrup was added to three times its volume ofwater with vigorous agitation. The white precipitate so obtained wasisolated by filtration and dried to yield 9.0 g. of polymer showing anintrinsic viscosity of 1.03 when dissolved in absolute methanol.

Discs of hydrophilic polymer, prepared as shaped articles from thissolution, measuring A1 inch in diameter and 0.05 mm. in thickness, weresaturated with nonpathogenic Fox strain poliomyelitis harvested inaqueous medium. The entrapped virus was then dried.

EXAMPLE 3 Into a flask equipped with an agitator and a heating mantlewas charged 1000 grams of silicone oil, 100 grams of 2-hydroxy ethylmethacrylate and 0.33 gram of isopropyl percarbonate. The fiask wasplaced under a nitrogen atmosphere and the contents were rapidlyagitated and heated to 100 C. After 15 minutes at 100 C., the polymerslurry obtained was filtered hot to isolate the polymer. The polymerpowder was reslurried in 300 ml. of xylene, filtered and dried.

In a separate container, 9.9 g. of 2-hydroxyethyl methacrylate is mixedwith 0.0214 gram of ethylene glycol dimethacrylate and 0.05 gram ofbenzoyl peroxide.

3.6 g. of the powder, when mixed with 9.9 g. of the formulatedhydroxyethyl methacrylate mixture formed a paste mixture. The mixturewas cast and cured to form a hollow cylinder 50 mm. long, with anoutside diameter of 5 mm. and walls 0.5 mm. thick. The hollow interiorof the cylinder was filled with rumen organisms and the hole sealed withfurther casting polymer.

Examples 4-7 illustrate other polymers suitable for use according to theinvention.

EXAMPLE 4 A solution was made of 100 parts of 2-hydroxyethyl acrylate,0.2 part of ethylene glycol dimethacrylate and 0.4 part of t-butylperoctoate and then cast into a mold and polymerized.

EXAMPLE 5 A solution was made of 100 parts of an isomeric mixture ofhydroxyisopropyl methacrylates, 0.2 part propylene glycoldirnethacrylate and 0.4 part of t-butyl peroctoate and then cast into amold and polymerized.

EXAMPLE 6 100 parts of Z-hydroxyethyl methacrylate was stirred with 0.05part of t-butyl peroctoate in a nitrogen atmosphere at a temperature of40 C. for 30 minutes. The resultant mixture was cooled to 25 C. andt-butyl peroctoate added so as to make the amount of t-butyl peroctoateadded in the system 0.15 part. 0.1 part of ethylene glycoldimethacrylate was also added along with the second addition of thet-butyl peroctoate and cast.

EXAMPLE 7 The process of Example 6 was repeated, substituting 0.2 partof 1,3-butylene glycol dimethacrylate in place of the ethylene glycoldimethacrylate as the cross-linking monomer.

EXAMPLE 8 100 parts of 2-hydroxyethyl methacrylate was stirred with 50parts of distilled water and 0.1 part of t-butyl peroctoate in ananaerobic atmosphere at a temperature of 40 C. for 20 minutes. The waterwas removed, alco hol added as a solvent and the resultant mixture wascooled to 25 C. and 0.05 part of t-butyl peroctoate added and at thesame time there was added 0.2 part of ethylene glycol dimethacrylate asa cross-linking monomer. The product was then polymerized to form asolution. There was aded L. casei and the mixture freeze dried.

EXAMPLE 9 Z-hydroxy ethyl methacrylate (100 g.) is mixed with tertiarybutyl peroctoate in the quantity 0.15 g./100 g. methacrylate. Ethyleneglycol dimethacrylate, in the concentration of 0.20 g./100 g. 2-hydroxyethyl methacrylate is added along with 1 gram of a foaming agent, sodiumbicarbonate. The mixture is heated to 70 C. and the resulting solid,friable polymeric foam is ground into fine powder of mesh. The polymericpowder so formed is mixed with attenuated poliomyelitis virus (Type 1(SM strain)) in aqueous fluid medium flavor solution and the resultantmixture is placed on a mechanical roller for approximately 2 hours. Thepolymeric powder thus absorbs the virus. The solution is then filteredand the residue freeze dried to form an entrapped virus in thehydrophilic polymer.

EXAMPLE 10 The process of the previous Example 9 is followed,substituting Saccharomyces cerevisiae for the polio virus.

EXAMPLE 11 2-hydroxy ethyl methacrylate g.) is mixed with tertiary butylperoctoate (0.20 g.). Ethylene glycol dimethacrylate (0.20 g.) is addedalong with 4 g. of a foaming agent, sodium bicarbonate. The mixture isheated to 70 C. and the resulting solid, friable polymeric foam isground into fine powder of 80 mesh. The polymeric powder formed is mixedwith 4% of aqueous polio virus Type I Sabin having an ID of 10' and theresultant mixture placed on a mechanical roller until the polymericpowder has absorbed the desired concentration of virus. The solution isthen filtered and the residue dried at 20 C. in vacuo.

EXAMPLE 12 The procedure of Example 11 was repeated using attenuatedI.B.R. virus (passed 50 times in lamb renal cells) and freeze dryingrather than drying at 20 C.

Similarly attenuated measles virus can be used to replace the I.B.R.virus as can B.C.G. vaccine.

EXAMPLE 13 40 lbs. of hydroxyethyl methacrylate, 4 lbs. of methacrylicacid, lbs. of methanol and 0.05 lb. of t-butyl peroctoate were heated to80 C. and stirred for 6 hours to elfect polymerization. To the'polymersolution was added slowly a 10-fold excess of acetone to precipitate thepolymer. After drying 36 lbs. of water soluble copolymer was obtained.

vThe polymer was dissolved in water to provide a 10 weight percentsolution. To the solution was added 1% of aqueous Saccharomycescerevisiae. The mixture was then cast as a 20-mil film on a polyethylenesheet and dried. The resulting brittle film was ground to- '60 mesh toyield a dry powder which dissolved and released the yeast upon contactwith water or milk.

In place of the S. cerevisiae there were also used L. casei, starterculture and I.B.R. virus to provide entrapped microorganisms, whichwerereleased on contact with aqueous liquid.

EXAMPLE 14 150 grams of distilled hydroxyethyl methacrylate, 600 gramsof methanol and 0.3 gram of t-butyl peroctoate (catalyst) arepolymerized in a three-neck flask equipped with a stirrer and a refluxcondenser, under a blanket of nitrogen, at 67 C. The resultinghydroxyethyl methacrylate polymer is then purified by slow precipitationin a tenfold excess of distilled water. The precipitated polymer is thenWashed thoroughly with distilled water, and dried overnight at roomtemperature under reduced pressure. (85% yield.)

EXAMPLE 15 A solution of 10 grams of the dry polymer of Example 14 in 85grams of ethylene glycol monomethyl ether was prepared and cooled to C.A solution of 0.2 gram ammonium dichromate in 5 ml. distilled water wasthen added and the solution was mixed during 5 minutes with a magneticstirrer to produce a cross-linkable hydroxyethyl methacrylate polymersolution.

EXAMPLE 16 A dispersion of 0.1 gram of yeast (S. cerevisiae) in 0.5 gramdistilled water was prepared, and mixed with 4.5 grams of the polymersolution prepared in Example 15.

On a horizontal casting table lined with poleythylene, a 8 mils thicklayer of polymer-yeast mixture was cast with a Gardner Casting Blade. Aflow of cold dry nitrogen was directed toward the surface of the filmduring 5 minutes. The film was then irradiated during more minutes witha General Electric U.V. Sunlamp (275 watts) placed 5 inches above thesurface to cross-link the polymer. A 0.5-mil thick transparent film wasobtained. On top of this film still positioned on the casting table, a2-mil thick layer of the solution of polyhydroxyethyl methacrylate asprepared in Example 2, was cast with the casting blade and immediatelyirradiated during 5 minutes with the U.V. Sunlamp. An insoluble bilayermembrane 0.6-rnil thick, with immobilized yeast therein, was obtained.The membrane consisted of the relatively thick film of cross-linkedhydroxyethyl methacrylate polymer containing entrapped urease and arelatively thin (0.1 mil) layer of cross-linked hydroxyethylmethacrylate polymer free of the yeast. The yeast containing layergenerally is at least twice as thick as the yeast free coating layer.The yeast containing layer for example can be from 0.25 mil to 10 milsor even more, e.g. 250 mils or higher.

A sample of yeast-containing film was introduced into a tube as shown inFIG. 3 and served as the intermediate section 30. A solution containing5-000 grams of sucrose in a liter of water was passed through the tube,entering the film on the yeast free side and emerging on the yeastcontaining side to form ethyl alcohol. The reaction was carried out at25 C.

EXAMPLE 17 In a flask, equipped with a magnetic stirrer, was prepared asolution containing 50 parts of hydroxyethyl methacrylate and 2 parts ofN,N-methylenebisacrylamide.

14 To this solution was added a dispersion of Rhizopus oryzae in 5 partsof water, and the contents of the flask were thoroughly mixed anddeaerated by bubbling nitrogen during 15 minutes. A solution of 0.25part of ammonium persulfate in 5 parts of water was then added to themixture, followed after one minute by a solution of 0.25 part of sodiumbisulfite in 5 parts of water. The contents of the flask wereimmediately poured in a mold formed by 2 glass plates separated by a 5mm. thick rubber gasket, positioned in a water bath at 10 C. Thepolymerization started immediately. After. 4 hours, the mold was openedand a strong foam-like polymeric gel entrapped R. oryzae was obtained.The thick gel slab was cut in irregular particles 2-3 mm. large. Theparticles were thoroughly washed with distilled water, and dried at 23C. under reduced pressure. They were then placed between two screen in apipe and a sugar solution containing 200 grams of sucrose per literpassed through the pipe. A large part of the sucrose in the solution,passed through the polymer particles, was converted to lactic acid. Theparticles of gel could also be coated with a 0.5- mil thick layer ofhydroxyethyl methacrylate polymer to enhance the shelf life of theentrapped R. oryzae containing particles.

EXAMPLE 18 The process of Example 17 was repeated, substitutingAspergillus niger for the R. oryzae and pouring the microorganismcontaining polymerizable mixture in glass tubes 0.8 cm. in diameter.Rods of A. niger entrapping gel were obtained. The rods were sliced intodiscs (25 mm. thick). The discs were coated on the top and bottom with acopolymer of hydroxyethyl methacrylate ethylene dimethacrylate(99.8:02). Each coating film was 1 mm. thick. A 10% aqueous sucrosesolution having a pH of 2.0 was passed through a tube as shown in FIG. 3having the coated disc as the intermediate section. The aqueous solutionemerging from the tube contained citric acid resulting from thefermentation of the sucrose.

EXAMPLE 19 10 g. hydroxyethyl methacrylate, 8 g. acrylamide, 5 g.methacrylic acid, 2 g. ethylene glycol dimethacrylate were mixed with 20g. of distilled water pH 7.0. A dispersion of Streptomyces humiaus in 35g. distilled water was added and the mixture was deaerated during 15minutes. A solution of 0.12 g. ammonium persulfate in 5 ml. water wasthen added while stirring, followed after one minute by a solution of0.12 g. of sodium bisulfite in 5 ml. water. The mixture was then pouredimmediately into a mold composed of the interval between the walls of 2concentric glass tubes, the larger of which had an internal diameter of4 mm. and the smaller an external diameter of 3 mm. in presence of aDacron mesh (5 mils) sheathing. The tube which was formed bypolymerizing is shown in FIG. 5. (To preserve the shelf life of themicroorganism the tube 18 can have inner and outer coatings of ahydrophilic water insoluble hydroxyethyl methacrylate polymer which doesnot contain microorganisms. To control the rate at which a material inthe lumen of the tube can reach the microorganism there can be used aninner coating of a hydrophilic Water insoluble hydroxyethyl methacrylatepolymer.)

In a specific example utilizing FIG. 5 an aqueous solution containing 3%corn steep liquor, 1% HCl-hydrolyzate of soybean meal, 0.1% K HPO 0.05%MgSO -7H O and sufficient Ca(OH) to give a pH of 7.0 was forced throughthe lumen of the tube. The solution diffused into the microorganismentrapped tube and dihydrostreptomycin diffused out of the outer surfaceof the external portion of the tube. The procedure was carried out at 28C.

Streptomycin can be prepared in similar manner employing Streptomycesgriseus as the microorganism and employing as the nutrient passedthrough the lumen of the tube in an aqueous composition having a pH of 7and 15 including per liter 15 grams of l-proline, 10 grams of glucose,grams of NaCl, 2 grams of K HPO 1 gram of MgSO -7H O, 0.4 gram of CaCl20 mg. of FeSO ,-7H O and 10 mg. of ZnSO -7H O.

What is claimed is:

1. A dosage consisting essentially of living therapeutic microorganismsentrapped or coated with a hydrophilic lower alkyl acrylate ormethacrylate polymer or a hydrophilic hydroxy lower alkoxy lower alkylacrylate or methacrylate polymer in dry form.

2. A dosage according to claim 1 wherein the hydrophilic polymer iswater soluble and is a polymer of hy droxyethyl acrylate, hydroxypropylacrylate, hydroxyethyl methacrylate or hydroxypropyl methacrylate.

3. A dosage according to claim 2 wherein the microorganism is entrappedin the polymer.

4. A dosage according to claim 1 wherein the hydrophilic polymer iswater insoluble and the therapeutic microorganisms are not elutabletherefrom.

5. A dosage according to claim 4 wherein the polymer is a polymer ofhydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylateor hydroxypropyl methacrylate.

6. A dosage according to claim 2 wherein there is an enteric coating onthe hydrophilic polymer entrapped therapeutic microorganism and thehydrophilic polymer is water soluble.

7. A dosage according to claim 2 wherein the therapeutic microorganismis a yeast.

8. A dosage according to claim 2 wherein the therapeutic microorganismis a bacteria.

9. A dosage according to claim 2 wherein the therapeutic microorganismis a virus.

10. A dosage according to claim 1 in the form of a dry powder.

11. A dosage according to claim 1 in which the dry microorganisms arepresent in a core surrounded by a coat of said polymer, said polymerbeing water insoluble.

12. A dry product comprising a solid water insoluble hydrophilic polymerof a member of the group consisting of hydroxy lower alkyl acrylates,hydroxy lower alkyl methacrylates, hydroxy lower alkoxy lower alkylacrylates and hydroxy lower alkoxy lower alkyl methacrylates, havingentrapped therein a living therapeutic or industrial microorganism.

13. A product according to claim 12 wherein the polymer is a sparinglycross-linked polymer.

14. A product according to claim 12 wherein the polymer is a polymer ofhydroxyethyl acrylate, hydroxyethyl methacrylate, hydroxypropyl acrylateor hydroxypropyl methacrylate.

15. A composition comprising a relatively thick layer of the product ofclaim 12 having on at least one surface thereof a relatively thinmembrane layer of a microorganism free water insoluble hydrophilicpolymer of a member of the group consisting of hydroxy lower alkylacrylates, hydroxy lower alkyl methacrylates, hydroxy lower alkoxy loweralkyl acrylates, hydroxy lower alkoxy lower alkyl methacrylates, vinylpyrrolidone, acrylamide, methacrylamide, N-lower alkyl acrylamide,N-lower alkyl methacrylamide, N-hydroxy lower alkyl acrylamide andN-hydroxy lower alkyl methacrylamide.

16. A composition according to claim 15 wherein the thick layer and thethin layer are both made of polymer of hydroxyethyl acrylate,hydroxyethyl methacrylate, hydroxypropyl acrylate or hydroxypropylmethacrylate.

17. A composition according to claim 16 wherein the thin membrane isfrom '5 to 500 microns.

18. A composition according to claim 17 wherein the thin membrane is1050 microns and the thick layer is at least twice as thick as said thinmembrane.

19. A composition according to claim 15 wherein the relatively thinmembrane layer is coated on one side only of said relatively thicklayer.

20. A composition according to claim 15 wherein the relatively thinlayer completely encases said relatively thick layer.

21. A product according to claim 12 in the form of a hollow tube.

22. A product according to claim 21 having reinforcing fibers embeddedtherein.

23. A tube according to claim 21 having a relatively thin overallinternal membrane coating of a microorganism free water insoluble,hydrophilic polymer of a member of the group consisting of hydroxy loweralkyl acrylates, hydroxy lower alkyl methacrylates, hydroxy lower alkoxylower alkyl acrylates, hydroxy lower alkoxy lower alkyl methacrylates,vinyl pyrrolidone, acrylamide, methacrylamide, N-lower alkyl acrylamide,N-lower alkyl rnethacrylamide, N-hydroxy lower alkyl acrylamide and Nhydroxy lower alkyl methacrylamide.

24. A product according to claim 12 wherein the hydrophilic polymer is acopolymer of said hydroxy alkyl aorylate or methacrylate or hydroxyalkoxyalkyl acrylate or methacrylate with up to 50% of a copolymerizableethylenically unsaturated monomer based on the weight of the monomericmixture.

25. A product according to claim 24 wherein the copolymerizable monomeris between 0.1 and 20% of said monomer mixture.

References Cited UNITED STATES PATENTS 3,046,201 7/1962 White et a1.195100 3,247,078 4/1966 Herrett 195102 3,577,512 5/1971 Shepherd et al.42481 X 3,220,960 11/1965 Wichterle et al. 2602.5 2,976,576 3/1961Wichterle et a1 18--58 SHEP K. ROSE, Primary Examiner U.S'. Cl. X. R.

UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. 3 767790 Dated OCtObQT 23 1973 Inventor(s) Alvin Guttag It is certified thaterror appears in the above-identified patent and that said LettersPatent are hereby corrected as shown below:

In the heading to the printed specification, lines 5 and 6, cancelContinuation-in-part of abandoned application Ser. No. 832, 938, June13, 1969.". Column 1, lines 19 and 20, cancel "This application is acontinuation-in-part of Ser. No. 832,938 filed June 13, 1969, nowabandoned."

Signed and sealed this 25th day of June 1974.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. C. MARSHALL DANN Attesting Officer Commissioner ofPatents ORM PC4050 uscoMM-Dc 60376-1 69 U.S. GOVERNMENT PRINTING OFFICE:i969 O-366-33A.

